EP3661950A2 - Gènes pesticides et leurs procédés d'utilisation - Google Patents

Gènes pesticides et leurs procédés d'utilisation

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Publication number
EP3661950A2
EP3661950A2 EP18755657.6A EP18755657A EP3661950A2 EP 3661950 A2 EP3661950 A2 EP 3661950A2 EP 18755657 A EP18755657 A EP 18755657A EP 3661950 A2 EP3661950 A2 EP 3661950A2
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EP
European Patent Office
Prior art keywords
toxin
plant
polypeptide
domain
pesticidal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18755657.6A
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German (de)
English (en)
Inventor
Jessica Parks
Kira Bulazel Roberts
Rebecca E. THAYER
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AgBiome Inc
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AgBiome Inc
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Filing date
Publication date
Application filed by AgBiome Inc filed Critical AgBiome Inc
Priority to EP22154386.1A priority Critical patent/EP4036105A3/fr
Priority to EP23170893.4A priority patent/EP4230642A2/fr
Publication of EP3661950A2 publication Critical patent/EP3661950A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention is drawn to methods and compositions for controlling pests, particularly plant pests.
  • Pests, plant diseases, and weeds can be serious threats to crops. Losses due to pests and diseases have been estimated at 37% of the agricultural production worldwide, with 13% due to insects, bacteria and other organisms.
  • Toxins are virulence determinants that play an important role in microbial pathogenicity and/or evasion of the host immune response.
  • Current strategies use the genes expressing these toxins to produce transgenic crops.
  • Transgenic crops expressing insecticidal protein toxins are used to combat crop damage from insects.
  • Bacillus toxins While the use of Bacillus toxins has been successful in controlling insects, resistance to Bt toxins has developed in some target pests in many parts of the world where such toxins have been used intensively.
  • One way of solving this problem is sowing Bt crops with alternating rows of regular non Bt crops (refuge).
  • An alternative method to avoid or slow down development of insect resistance is stacking insecticidal genes with different modes of action against insects in transgenic plants.
  • the current strategy of using transgenic crops expressing insecticidal protein toxins is placing increasing emphasis on the discovery of novel toxins, beyond those already derived from the bacterium Bacillus thuringiensis. These toxins may prove useful as alternatives to those derived from B. thuringiensis for deployment in insect- and pest-resistant transgenic plants. Thus, new toxin proteins are needed.
  • compositions having pesticidal activity and methods for their use are provided.
  • compositions include isolated and recombinant polypeptide sequences having pesticidal activity, recombinant and synthetic nucleic acid molecules encoding the pesticidal polypeptides, DNA constructs comprising the nucleic acid molecules, vectors comprising the nucleic acid molecules, host cells comprising the vectors, and antibodies to the pesticidal polypeptides.
  • Nucleotide sequences encoding the polypeptides provided herein can be used in DNA constructs or expression cassettes for transformation and expression in organisms of interest, including microorganisms and plants.
  • compositions and methods provided herein are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes.
  • Transgenic plants and seeds comprising a nucleotide sequence that encodes a pesticidal protein of the invention are also provided. Such plants are resistant to insects and other pests.
  • Methods are provided for producing the various polypeptides disclosed herein, and for using those polypeptides for controlling or killing a pest. Methods and kits for detecting polypeptides of the invention in a sample are also included.
  • compositions and method for conferring pesticidal activity to an organism are provided.
  • the modified organism exhibits pesticidal resistance or tolerance.
  • Recombinant pesticidal proteins, or polypeptides and fragments and variants thereof that retain pesticidal activity are provided and include those set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91
  • the pesticidal proteins are biologically active (e.g., pesticidal) against pests including insects, fungi, nematodes, and the like.
  • Nucleotides encoding the pesticidal polypeptides including for example, SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
  • the pesticidal proteins are biologically active (for example, are pesticidal) against pests including insects, fungi, nematodes, and the like.
  • the pesticidal polypeptides and the active variant and fragments thereof have an improved pesticidal activity when compared to other polypeptides in the art.
  • Polynucleotides encoding the pesticidal polypeptides including for example, SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
  • the transformed organisms are characterized by genomes that comprise at least one stably incorporated DNA construct comprising a coding sequence for a pesticidal protein disclosed herein.
  • the coding sequence is operably linked to a promoter that drives expression of the encoded pesticidal polypeptide. Accordingly, transformed microorganisms, plant cells, plant tissues, plants, seeds, and plant parts are provided. A summary of various polypeptides, active variants and fragments thereof, and polynucleotides encoding the same are set forth below in Table 1. As noted in Table 1, various forms of polypeptides are provided. Full length pesticidal polypeptides, as well as, modified versions of the original full-length sequence (i.e., variants) are provided.
  • CryBPl sequences Such sequences (SEQ ID NOS: 8, 100, and 289) comprise accessory polypeptides that can be associated with some of the toxin genes. In such instances, the CryBPl sequences can be used alone or in combination with any of the pesticidal polypeptides provided herein. Such sequence comprise the sequence of a downstream protein that has homology to the C-terminal end of the Cry class of toxin genes and are usually found after a Cry gene that is not full-length and is missing the expected C-terminal region.
  • APG00557.0 (39.39% identity, 49.81% similarity)
  • APG00706.0 (26.62% identity, 36.95% similarity)
  • the pesticidal proteins provided herein and the nucleotide sequences encoding them are useful in methods for impacting pests. That is, the compositions and methods of the invention find use in agriculture for controlling or killing pests, including pests of many crop plants.
  • the pesticidal proteins provided herein are toxin proteins from bacteria and exhibit activity against certain pests.
  • the pesticidal proteins are from several classes of toxins including Cry, Cyt, BIN, Mtx toxins. See, for example, Table 1 for the specific protein classifications of the various SEQ ID NOS provided herein.
  • Pfam database entries The Pfam database is a database of protein families, each represented by multiple sequence alignments and a profile hidden Markov model. Finn et al. (2014) Nucl. Acid Res.
  • Bacillus thuringiensis is a gram-positive bacterium that produces insecticidal proteins as crystal inclusions during its sporulation phase of growth.
  • the proteinaceous inclusions of Bacillus thuringiensis (Bt) are called crystal proteins or ⁇ -endotoxins (or Cry proteins), which are toxic to members of the class Insecta and other invertebrates.
  • Cyt proteins are parasporal inclusion proteins from Bt that exhibits hemolytic (Cytolitic) activity or has obvious sequence similarity to a known Cyt protein. These toxins are highly specific to their target organism, are innocuous to humans, vertebrates, and plants.
  • the structure of the Cry toxins reveals five conserved amino acid blocks, concentrated mainly in the center of the domain or at the junction between the domains.
  • the Cry toxin consists of three domains, each with a specific function. Domain I is a seven a-helix bundle in which a central helix is completely surrounded by six outer helices. This domain is implicated in channel formation in the membrane. Domain II appears as a triangular column of three anti-parallel ⁇ -sheets, which are similar to antigen-binding regions of immunoglobulins. Domain III contains anti-parallel ⁇ -strands in a ⁇ sandwich form.
  • the N-terminal part of the toxin protein is responsible for its toxicity and specificity and contains five conserved regions. The C-terminal part is usually highly conserved and probably responsible for crystal formation. See, for example, U.S. Patent No. 8,878,007.
  • cry proteins have been classified into groups based on toxicity to various insect and invertebrate groups.
  • Cry I demonstrates toxicity to lepidopterans, Cry II to lepidopterans and dipterans, Crylll to coleopterans, Cry IV to dipterans, and Cry V and Cry VI to nematodes.
  • New Cry proteins can be identified and assigned to a Cry group based on amino acid identity. See, for example, Bravo, A.
  • cry gene family consists of several phylogentically non-related protein families that may have different modes of action: the family of three-domain Cry toxins, the family of mosquitocidal Cry toxins, the family of the binary-like toxins, and the Cyt family of toxins (Bravo et al., 2005). Some Bt strains produce additional insecticidal toxins, the VIP toxins. See, also, Cohen et al. (2011) J. Mol. Biol. 413:4-814; Crickmore et al. (2014) Bacillus thuringiensis toxin nomenclature, found on the world wide web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/;
  • Cyt designates a parasporal crystal inclusion protein from Bacillus thuringiensis with cytolytic activity, or a protein with sequence similarity to a known Cyt protein. (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62: 807-813). The gene is denoted by cyt. These proteins are different in structure and activity from Cry proteins (Gill et al. (1992) Annu. Rev. Entomol. 37: 615-636). The Cyt toxins were first discovered in B. thuringiensis subspecies israelensis (Goldberg et al. (1977) Mosq. News. 37: 355-358).
  • Cyt2A The structure of Cyt2A, solved by X-ray crystallography, shows a single domain where two outer layers of a-helix wrap around a mixed ⁇ -sheet. Further available crystal structures of Cyt toxins support a conserved ⁇ - ⁇ structural model with two a-helix hairpins flanking a ⁇ -sheet core containing seven to eight ⁇ -strands. (Cohen et al. (2011) J. Mol. Biol. 413: 80 4-814) Mutagenic studies identified ⁇ - sheet residues as critical for toxicity, while mutations in the helical domains did not affect toxicity (Adang et al.; Diversity of Bacillus thuringiensis Crystal Toxins and Mechanism of Action. In: T. S. Dhadialla and S.
  • Cyt toxin The representative domain of the Cyt toxin is a ⁇ -endotoxin, Bac_thur_toxin (Pfam PF01338).
  • CytlA Cyt proteins
  • CytlA and Cyt2A protoxins are processed by digestive proteases at the same sites in the N- and C-termini to a stable toxin core. Cyt toxins then interact with non-saturated membrane lipids, such as phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin.
  • Cyt toxins For Cyt toxins, pore-formation and detergent-like membrane disruption have been proposed as nonexclusive mechanisms; and it is generally accepted that both may occur depending on toxin concentration, with lower concentrations favoring oligomeric pores and higher concentrations leading to membrane breaks. (Butko (2003) Appl. Environ. Microbiol. 69: 2415-2422) In the pore-formation model, the Cyt toxin binds to the cell membrane, inducing the formation of cation-selective channels in the membrane vesicles leading to colloid-osmotic lysis of the cell. (Knowles et al. (1989) FEBS Lett. 244: 259-262;
  • a number of pesticidal proteins unrelated to the Cry proteins are produced by some strains of B. thuringiensis and B. cereus during vegetative growth (Estruch et al. (1996) Proc Natl Acad Sci USA 93:5389-5394; Warren et al. (1994) WO 94/21795). These vegetative insecticidal proteins, or Vips, do not form parasporal crystal proteins and are apparently secreted from the cell. The Vips are presently excluded from the Cry protein nomenclature because they are not crystal-forming proteins.
  • the term VIP is a misnomer in the sense that some B.
  • thuringiensis Cry proteins are also produced during vegetative growth as well as during the stationary and sporulation phases, most notably Cry3 Aa.
  • the location of the Vip genes in the B. thuringiensis genome has been reported to reside on large plasmids that also encode cry genes (Mesrati et al. (2005) FEMS Microbiol. Lett. 244(2):353-8).
  • a web-site for the nomenclature of Bt toxins can be found on the world wide web at lifesci.sussex.ac.uk with the path
  • Vip genes form binary two-component protein complexes; an "A” component is usually the “active” portion, and a “B” component is usually the "binding" portion.
  • Vipl and Vip4 proteins generally contain binary toxin B protein domains.
  • Vip2 proteins generally contain binary toxin A protein domains.
  • Vipl and Vip2 proteins are the two components of a binary toxin that exhibits toxicity to coleopterans.
  • ViplAal and Vip2Aal are very active against corn rootworms, particularly Diabrotica virgifera and Diabrotica longicornis (Han et al.
  • NAD-dependent ADP-ribosyltransferase Vip2 likely modifies monomeric actin at Argl77 to block polymerization, leading to loss of the actin cytoskeleton and eventual cell death due to the rapid subunit ex-change within actin filaments in vivo (Carrier M. F. (1990) Adv. Biophys. 26:51-73).
  • Vip3A toxins are pore-forming proteins capable of making stable ion channels in the membrane (Lee et al. (2003) Appl. Environ. Microbiol. 69:4648-4657). Vip3 proteins are active against several major lepidopteran pests (Rang et al. (2005) Appl. Environ. Microbiol. 71(10):6276-6281; Bhalla et al. (2005) FEMS Microbiol. Lett. 243:467-472; Estruch et al. (1998) WO 9844137; Estruch et al. (1996) Proc Natl Acad Sci USA 93:5389-5394; Selvapandiyan et al. (2001) Appl. Environ Microbiol. 67:5855-5858; Yu et al. (1997) Appl. Environ Microbiol. 63:532-536).
  • Vip3A is active against Agrotis ipsilon, Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens, and Helicoverpa zea (Warren et al. (1996) WO 96/10083; Estruch et al. (1996) Proc Natl Acad Sci USA 93:5389-5394).
  • Vip3A proteins must be activated by proteases prior to recognition at the surface of the midgut epithelium of specific membrane proteins different from those recognized by Cry toxins.
  • the MTX family of toxin proteins is characterized by the presence of a conserved domain, ETX_MTX2 (pfam 03318).
  • ETX_MTX2 pfam 033128.
  • Members of this family share sequence homology with the mosquitocidal toxins Mtx2 and Mtx3 from Bacillus sphaericus, as well as with the epsilon toxin ETX from Clostridium perfringens (Cole et al. (2004) Nat. Struct. Mol. Biol. 11: 797-8; Thanabalu et al. (1996) Gene 170:85-9).
  • the MTX-like proteins are structurally distinct from the three-domain Cry toxins, as they have an elongated and predominately ⁇ -sheet-based structure.
  • the MTX-like proteins are thought to form pores in the membranes of target cells (Adang et al. (2014) supra). Unlike the three-domain Cry proteins, the MTX-like proteins are much smaller in length, ranging from 267 amino acids (Cry23) to 340 amino acids (Cryl5A.
  • the members of the MTX-like toxin family include Cryl5, Cry23, Cry33, Cry38, Cry45, Cry46, Cry51, Cry60A, Cry60B, and Cry64.
  • This family exhibits a range of insecticidal activity, including activity against insect pests of the Lepidopteran and Coleopteran orders. Some members of this family may form binary partnerships with other proteins, which may or may not be required for insecticidal activity.
  • Cry 15 is a 34 kDA protein that was identified in Bacillus thuringiensis serovar thompsoni HD542; it occurs naturally in a crystal together with an unrelated protein of approximately 40 kDa.
  • the gene encoding Cry 15 and its partner protein are arranged together in an operon.
  • Cry 15 alone has been shown to have activity against lepidopteran insect pests including Manduca sexta, Cydia pomonella, and Pieris rapae, with the presence of the 40 kDA protein having been shown to increase activity of Cry 15 only against C. pomonella (Brown K. and Whiteley H. (1992) J. Bacteriol. 174:549-557; Naimov et al. (2008) Appl. Environ.
  • Cry23 is a 29 kDA protein that has been shown to have activity against the coleopteran pests Tribolium castaneum and Popillia japonica together with its partner protein Cry37 (Donovan et al. ( 2000) US Patent No. 6,063,756).
  • An ETX_MTX toxin gene was recently identified in the genome of Bacillus thuringiensis serovar tolworthi strain Na205-3. This strain was found to be toxic against the lepidpoteran pest Helicoverpa armigera, and it also contained homologs of Cryl, Cry 11, Vipl, Vip2, and Vip3 (Palma et al. (2014) Genome Announc. 2(2): e00187-14. Published online Mar 13, 2014 at doi: 10.1128/genomeA.00187-14; PMCID: PMC3953196).
  • the MTX-like proteins have a unique domain structure relative to the three- domain Cry proteins, they are believed to possess a unique mode of action, thereby making them a valuable tool in insect control and the fight against insect resistance.
  • Bacterial cells produce large numbers of toxins with diverse specificity against host and non-host organisms. Large families of binary toxins have been identified in numerous bacterial families, including toxins that have activity against insect pests.
  • Ls Lysinibacillus sphaericus
  • Bacillus sphaericus Bacillus sphaericus
  • BinA/BinB This binary complex forms a parasporal crystal in Ls cells and has strong and specific activity against dipteran insects, specifically mosquitos. In some areas, insect resistance to existing Ls mosquitocidal strains has been reported. The discovery of new binary toxins with different target specificity or the ability to overcome insect resistance is of significant interest.
  • the Ls binary insecticidal protein complex contains two major polypeptides, a 42 kDa polypeptide and a 51 kDa polypepdide, designated BinA and BinB, respectively (Ahmed et al. (2007) supra).
  • the two polypeptides act synergistically to confer toxicity to their targets. Mode of action involves binding of the proteins to receptors in the larval midgut. In some cases, the proteins are modified by protease digestion in the larval gut to produce activated forms.
  • the BinB component is thought to be involved in binding, while the BinA component confers toxicity (Nielsen-LeRoux et al. (2001) Appl. Environ. Microbiol. 67(l l):5049-5054). When cloned and expressed separately, the BinA component is toxic to mosquito larvae, while the BinB component is not. However, coadministration of the proteins markedly increases toxicity (Nielsen-LeRoux et al. (2001) supra).
  • Bin protein homologs have been described from bacterial sources. Priest et al. (1997) Appl. Environ. Microbiol. 63(4): 1195-1198 describe a hybridization effort to identify new Ls strains, although most of the genes they identified encoded proteins identical to the known BinA/BinB proteins.
  • the BinA protein contains a defined conserved domain known as the Toxin 10 superfamily domain. This toxin domain was originally defined by its presence in BinA and BinB. The two proteins both have the domain, although the sequence similarity between BinA and BinB is limited in this region ( ⁇ 40%).
  • the Cry49Aa protein which also has insecticidal activity, also has this domain (described below).
  • the Cry48Aa/Cry49Aa binary toxin of Ls has the ability to kill Culex
  • quinquefasciatus mosquito larvae are in a protein structural class that has some similarity to the Cry protein complex of Bacillus thuringiensis (Bt), a well-known insecticidal protein family.
  • Bt Bacillus thuringiensis
  • the Cry34/Cry35 binary toxin of Bt is also known to kill insects, including Western corn rootworm, a significant pest of corn.
  • Cry34, of which several variants have been identified, is a small (14 kDa) polypeptide
  • Cry35 also encoded by several variants
  • Cry35 is a 44 kDa polypeptide.
  • Phosphoinositide phospholipase C proteins are members of the broader group of phospholipase C proteins. Many of these proteins play important roles in signal transduction as part of normal cell physiology. Several important bacterial toxins also contain domains with similarity to these proteins (Titball, R.W. (1993) Microbiological Reviews. 57(2):347-366).
  • the PI-PLC toxin class occurs in Bacillus isolates, commonly seen in cooccurrence with homologs to other described toxin classes, such as Binary Toxins.
  • This class of sequences has homology to phosphatidylinositol phosphodiesterases (also referred to as phosphatidylinositol- specific phospholipase C - PI-PLC).
  • the crystal structure and its active site were solved for B. cereus PI-PLC by Heinz et al (Heinz, et. al, (1995) The EMBO Journal. 14(16): 3855-3863). The roles of the B.
  • cereus PI-PLC active site amino acid residues in catalysis and substrate binding were investigated by Gassier et al using site-directed mutagenesis, kinetics, and crystal structure analysis (Gassier, et. al, (1997) Biochemistry. 36(42): 12802-13).
  • PI-PLC toxin proteins contain a PLC-like phosphodiesterase, TIM beta/alpha-barrel domain (IPR017946) and/or a Phospholipase C, phosphatidylinositol- specific, X domain (IPR000909) (also referred to as the PI-PLC X-box domain).
  • TIM beta/alpha-barrel domain IPR017946
  • Phospholipase C phosphatidylinositol- specific, X domain
  • IPR000909 also referred to as the PI-PLC X-box domain
  • This list includes most commonly a lectin domain (IPR000772), a sugar- binding domain that can be present in one or more copies and is thought to bind cell membranes, as well as the Insecticidal crystal toxin (IPR008872) (also referred to as ToxinlO or P42), which is the defining domain of the Binary Toxin.
  • IPR008872 Insecticidal crystal toxin
  • toxins of this PI-PLC class were defined in U.S. Patent No. 8,318,900 B2 SEQ ID NOs 30 (DNA) and 79 (amino acid), in U.S. Patent Publication No.
  • pesticidal proteins from these classes of toxins.
  • the pesticidal proteins are classified by their structure, homology to known toxins and/or their pesticidal specificity. ii. Variants and Fragments of Pesticidal Proteins and Polynucleotides Encoding the Same
  • Pesticidal proteins or polypeptides of the invention include those set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
  • pesticidal toxin or “pesticidal protein” or “pesticidal polypeptide” is intended a toxin or protein or polypeptide that has activity against one or more pests, including, insects, fungi, nematodes, and the like such that the pest is killed or controlled.
  • an "isolated” or “purified” polypeptide or protein, or biologically active portion thereof is substantially or essentially free from components that normally accompany or interact with the polypeptide or protein as found in its naturally occurring environment.
  • an isolated or purified polypeptide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.
  • optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • fragment refers to a portion of a polypeptide sequence of the invention.
  • “Fragments” or “biologically active portions” include polypeptides comprising a sufficient number of contiguous amino acid residues to retain the biological activity, i.e., have pesticidal activity. Fragments of the pesticidal proteins include those that are shorter than the full-length sequences, either due to the use of an alternate downstream start site, or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism the protein is expressed in, or in the pest after ingestion of the protein. Examples of fragments of the proteins can be found in Table 1.
  • a biologically active portion of a pesticidal protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250 or more amino acids in length of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101
  • a fragment comprises at least 8 contiguous amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103
  • Bacterial genes including those encoding the pesticidal proteins disclosed herein, quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. On rare occasions, translation in bacterial systems can initiate at a TTG codon, though in this event the TTG encodes a methionine.
  • the pesticidal proteins provided herein include amino acid sequences deduced from the full-length nucleotide sequences and amino acid sequences that are shorter than the full-length sequences due to the use of an alternate downstream start site.
  • the nucleotide sequence of the invention and/or vectors, host cells, and plants comprising the nucleotide sequence of the invention may comprise a nucleotide sequence encoding an alternate start site.
  • modifications may be made to the pesticidal polypeptides provided herein creating variant proteins. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques. Alternatively, native, as yet-unknown or as yet unidentified polynucleotides and/or polypeptides structurally and/or functionally-related to the sequences disclosed herein may also be identified that fall within the scope of the present invention. Conservative amino acid substitutions may be made in nonconserved regions that do not alter the function of the pesticidal proteins. Alternatively, modifications may be made that improve the activity of the toxin.
  • domain III swapping Modification of Cry toxins by domain III swapping has resulted in some cases in hybrid toxins with improved toxicities against certain insect species.
  • domain III swapping could be an effective strategy to improve toxicity of Cry toxins or to create novel hybrid toxins with toxicity against pests that show no susceptibility to the parental Cry toxins.
  • Site-directed mutagenesis of domain II loop sequences may result in new toxins with increased insecticidal activity.
  • Domain II loop regions are key binding regions of initial Cry toxins that are suitable targets for the mutagenesis and selection of Cry toxins with improved insecticidal properties.
  • Domain I of the Cry toxin may be modified to introduce protease cleavage sites to improve activity against certain pests. Strategies for shuffling the three different domains among large numbers of cry genes and high through output bioassay screening methods may provide novel Cry toxins with improved or novel toxicities.
  • Pesticidal activity comprises the ability of the composition to achieve an observable effect diminishing the occurrence or an activity of the target pest, including for example, bringing about death of at least one pest, or a noticeable reduction in pest growth, feeding, or normal physiological development.
  • Such decreases in numbers, pest growth, feeding or normal development can comprise any statistically significant decrease, including, for example a decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater.
  • pesticidal activity against one or more of the various pests including, for example, pesticidal activity against Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Nematodes,
  • Thysanoptera Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., or any other pest described herein. It is recognized that the pesticidal activity may be different or improved relative to the activity of the native protein, or it may be unchanged, so long as pesticidal activity is retained. Methods for measuring pesticidal activity are provide elsewhere herein. See also,Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein
  • variants polypeptides having an amino acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
  • a biologically active variant of a pesticidal polypeptide of the invention may differ by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6- 10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue.
  • the polypeptides can comprise an N-terminal or a C-terminal truncation, which can comprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50 amino acids or more from either the N or C terminal of the polypeptide.
  • the active variant comprising any one of SEQ ID NOs: 1-393 can comprise at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
  • the active variant will comprise at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: l, and further comprises the native amino acids at positions 209-398.
  • APG01052.1 15 Alternate PF05791 Bacillus haemolytic enterotoxin 82 210 start (H BL)
  • APG01135.0 20 PF03945 delta endotoxin, N-terminal 51 300 domain
  • APG01261.0 24 PF03945 delta endotoxin, N-terminal 72 311 domain
  • APG01446.0 35 PF03945 delta endotoxin, N-terminal 133 358 domain
  • APG01446.3 38 Signal PF03945 delta endotoxin, N-terminal 85 310 peptide domain
  • APG01679.1 41 Signal PF03945 delta endotoxin, N-terminal 81 260 peptide domain
  • APG01874.1 50 Alternate PF03318 Clostridium epsilon toxin 82 291 start ETX/Bacillus mosquitocidal toxin
  • APG02007.1 58 Signal PF03318 Clostridium epsilon toxin 54 264 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG02242.0 68 PF03945 delta endotoxin, N-terminal 68 306 domain
  • APG02401.1 74 Signal PF03318 Clostridium epsilon toxin 62 281 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG02801.1 Signal PF03318 Clostridium epsilon toxin 65 284 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG03223.1 104 Signal PF05431 Insecticidal Crystal Toxin, P42 178 370 peptide
  • APG03444.1 106 Signal PF03318 Clostridium epsilon toxin 77 284 peptide ETX/Bacillus mosquitocidal toxin removed MTX2 APG03497.0 107 PF05431 Insecticidal Crystal Toxin, P42 144 329
  • APG03506.1 109 Signal PF03318 Clostridium epsilon toxin 56 267 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG03638.1 116 Signal PF03318 Clostridium epsilon toxin 68 206 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG04342.1 130 Signal PF03318 Clostridium epsilon toxin 92 204 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG04479.2 135 Signal PF03318 Clostridium epsilon toxin 64 268 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG04531.1 137 Signal PF03318 Clostridium epsilon toxin 60 264 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG04536.0 138 PF 14200 Ricin-type beta-trefoil lectin 5 96 domain-like PF05431 Insecticidal Crystal Toxin, P42 150 342
  • APG04689.1 Alternate PF03945 delta endotoxin, N-terminal 74 262 start domain
  • APG04689.2 Alternate PF03945 delta endotoxin, N-terminal 74 262 start and 3' domain
  • APG04789.1 152 Signal PF03318 Clostridium epsilon toxin 56 259 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG05139.1 165 Signal PF03945 delta endotoxin, N-terminal 82 257 peptide domain
  • APG05424.1 179 Alternate PF03945 delta endotoxin, N-terminal 69 295 start domain
  • APG05439.0 180 PF03945 delta endotoxin, N-terminal 141 322 domain
  • APG05439.1 181 Alternate PF03945 delta endotoxin, N-terminal 124 305 start domain
  • APG05468.1 184 Signal PF03318 Clostridium epsilon toxin 64 264 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG05632.0 190 PF07523 Bacterial Ig-like domain (group 295 346
  • APG05632.1 191 Alternate PF07523 Bacterial Ig-like domain (group 289 340 start 3)
  • APG05632.2 192 Signal PF07523 Bacterial Ig-like domain (group 261 312 peptide 3)
  • APG06056.0 204 PF03945 delta endotoxin, N-terminal 68 275 domain
  • APG06121.1 207 Alternate PF01338 Bacillus thuringiensis toxin 3 188 start
  • APG06156.1 210 Alternate PF03945 delta endotoxin, N-terminal 56 274 start and 3' domain
  • APG06452.1 215 Alternate PF03318 Clostridium epsilon toxin 107 307 start ETX/Bacillus mosquitocidal toxin
  • APG06452.2 216 Alternate PF03318 Clostridium epsilon toxin 52 252 start ETX/Bacillus mosquitocidal toxin
  • APG06456.1 218 Alternate PF03945 delta endotoxin, N-terminal 59 292 start and 3' domain
  • APG06635.1 223 Alternate PF03318 Clostridium epsilon toxin 50 168 start ETX/Bacillus mosquitocidal toxin
  • APG06681.2 226 Alternate PF03945 delta endotoxin, N-terminal 72 340 start and 3' domain
  • APG06729.1 228 Signal PF03318 Clostridium epsilon toxin 72 304 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG06744.1 Alternate PF03318 Clostridium epsilon toxin 65 283 start ETX/Bacillus mosquitocidal toxin
  • APG06744.2 231 Signal PF03318 Clostridium epsilon toxin 39 257 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG06751.1 Alternate PF03945 delta endotoxin, N-terminal 108 263 start domain
  • APG06751.2 234 Signal PF03945 delta endotoxin, N-terminal 65 220 peptide domain
  • APG06820.1 236 Alternate PF03318 Clostridium epsilon toxin 135 272 start ETX/Bacillus mosquitocidal toxin
  • APG06820.2 237 Signal PF03318 Clostridium epsilon toxin 106 243 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG07047.1 246 Alternate PF03945 delta endotoxin, N-terminal 59 319 start and 3' domain
  • APG07202.1 250 Alternate PF14200 Ricin-type beta-trefoil lectin 48 144 start domain-like
  • APG07340.1 252 Alternate PF06101 Plant protein of unknown 4 160 start function (DUF946)
  • APG07340.2 253 Alternate PF06101 Plant protein of unknown 4 170 start function (DUF946)
  • APG07707.1 260 Alternate PF03945 delta endotoxin, N-terminal 123 314 start domain APG07707.2 261 Signal PF03945 delta endotoxin, N-terminal 87 278 peptide domain
  • APG07751.1 263 Alternate PF03945 delta endotoxin, N-terminal 65 307 start domain
  • APG08265.0 271 PF03945 delta endotoxin, N-terminal 36 257 domain
  • APG08298.1 273 Alternate PF03318 Clostridium epsilon toxin 33 270 start ETX/Bacillus mosquitocidal toxin
  • APG08366.0 276 PF05791 Bacillus haemolytic enterotoxin 69 199 (H BL)
  • APG08414.1 278 Alternate PF07691 PA14 domain 45 170 start
  • APG08601.0 279 PF03945 delta endotoxin, N-terminal 218 383 domain
  • APG08601.1 280 Alternate PF03945 delta endotoxin, N-terminal 122 287 start domain
  • APG08601.2 281 Signal PF03945 delta endotoxin, N-terminal 84 249 peptide domain
  • APG09469.1 307 Alternate PF03318 Clostridium epsilon toxin 115 341 start ETX/Bacillus mosquitocidal toxin
  • APG09469.2 308 Signal PF03318 Clostridium epsilon toxin 84 310 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG09668.0 315 PF03945 delta endotoxin, N-terminal 57 296 domain
  • APG09719.1 317 Alternate PF03318 Clostridium epsilon toxin 134 340 start ETX/Bacillus mosquitocidal toxin
  • APG09719.2 Signal PF03318 Clostridium epsilon toxin 109 315 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG09794.1 324 Signal PF03945 delta endotoxin, N-terminal 108 323 peptide domain
  • APG09794.2 325 Signal PF03945 delta endotoxin, N-terminal 108 323 peptide domain
  • APG01045.0 332 PF03945 delta endotoxin, N-terminal 90 314 domain PF00555 delta endotoxin 322 513
  • APG03553.1 338 Signal PF03318 Clostridium epsilon toxin 199 327 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG04702.1 341 Signal PF03318 Clostridium epsilon toxin 64 264 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG05105.1 344 Alternate PF03945 delta endotoxin, N-terminal 107 337 start domain
  • APG05105.2 345 Signal PF03945 delta endotoxin, N-terminal 77 307 peptide domain
  • APG05105.3 346 Alternate PF03945 delta endotoxin, N-terminal 107 337 start and 3' domain
  • APG05105.4 347 Signal PF03945 delta endotoxin, N-terminal 77 307 peptide domain
  • APG05140.1 349 Alternate PF03318 Clostridium epsilon toxin 113 303 start ETX/Bacillus mosquitocidal toxin
  • APG05183.1 351 Signal PF03318 Clostridium epsilon toxin 54 263 peptide ETX/Bacillus mosquitocidal toxin removed MTX2
  • APG05689.1 354 Alternate PF03945 delta endotoxin, N-terminal 122 304 start domain
  • APG05689.2 355 Signal PF03945 delta endotoxin, N-terminal 84 266 peptide domain
  • APG07291.1 357 Signal PF03945 delta endotoxin, N-terminal 81 254 peptide domain

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Abstract

La présente invention concerne des compositions présentant une activité pesticide et leurs procédés d'utilisation. Les compositions comprennent des séquences polypeptidiques isolées et de recombinaison présentant une activité pesticide, des molécules d'acides nucléiques de recombinaison et de synthèse codant pour les polypeptides pesticides, des constructions d'ADN comprenant les molécules d'acides nucléiques, des vecteurs comprenant les molécules d'acides nucléiques, des cellules hôtes comprenant les vecteurs et des anticorps dirigés contre les polypeptides pesticides. Des séquences nucléotidiques codant pour les polypeptides peuvent être utilisées dans des constructions ou dans des cassettes d'expression d'ADN en vue d'une transformation et d'une expression dans des organismes d'intérêt, notamment des micro-organismes et des plantes. Les compositions et les procédés selon l'invention sont utiles pour la production d'organismes présentant une résistance ou une tolérance accrue aux parasites. L'invention concerne également des semences et des plantes transgéniques comprenant une séquence nucléotidique qui code pour une protéine pesticide selon l'invention. De telles plantes sont résistantes aux insectes et autres parasites. L'invention concerne en outre des procédés de production des divers polypeptides selon l'invention, et d'utilisation de ces polypeptides pour lutter contre un parasite ou le tuer.
EP18755657.6A 2017-08-03 2018-08-02 Gènes pesticides et leurs procédés d'utilisation Withdrawn EP3661950A2 (fr)

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PY1864301A (es) 2021-06-18
US20190040411A1 (en) 2019-02-07
CN111247164A (zh) 2020-06-05
EP4036105A3 (fr) 2022-11-02
EP4036105A2 (fr) 2022-08-03
US20240052364A1 (en) 2024-02-15
US20210010019A1 (en) 2021-01-14
CA3071862A1 (fr) 2019-02-07
WO2019028235A3 (fr) 2019-03-14
WO2019028235A2 (fr) 2019-02-07
BR112020002279A2 (pt) 2020-07-28

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